pcr (version 1.2.2)

pcr_efficiency: Calculate amplification efficiency

Description

Uses the \(C_T\) values from a serial dilution experiment to calculate the amplification efficiency of a PCR reaction.

Usage

pcr_efficiency(df, amount, reference_gene, plot = FALSE)

Arguments

df

A data.frame of \(C_T\) values with genes in the columns and samples in rows rows. Each sample are replicates of a known input/dilution given by amount

amount

A numeric vector of the input amounts or dilutions. The length of this vector should equal the row number of df

reference_gene

A character string of the column name of a control gene

plot

A logical (default FALSE) to indicate whether to return a data.frame or a plot

Value

When plot is FALSE returns a data.frame of 4 columns describing the line between the \(\Delta C_T\) of target genes and the log of amount

• gene The column names of df. reference_gene is dropped

• intercept The intercept of the line

• slope The slope of the line

• r_squared The squared correlation

When plot is TRUE returns a graph instead shows the average and standard deviation of of the \(\Delta C_T\) at different input amounts. In addition, a linear trend line is drawn.

Details

Fortunately, regardless of the method used in the analysis of qPCR data, The quality assessment are done in a similar way. It requires an experiment similar to that of calculating the standard curve. Serial dilutions of the genes of interest and controls are used as input to the reaction and different calculations are made. The amplification efficiency is approximated be the linear trend between the difference between the \(C_T\) value of a gene of interest and a control/reference (\(\Delta C_T\)) and the log input amount. This piece of information is required when using the \(\Delta \Delta C_T\) model. Typically, the slope of the curve should be very small and the \(R^2\) value should be very close to one. Other analysis methods are recommended when this is not the case.

References

Livak, Kenneth J, and Thomas D Schmittgen. 2001. <U+201C>Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the Double Delta CT Method.<U+201D> Methods 25 (4). ELSEVIER. doi:10.1006/meth.2001.1262.

Examples

# NOT RUN {
# locate and read file
fl <- system.file('extdata', 'ct3.csv', package = 'pcr')
ct3 <- read.csv(fl)

# make amount/dilution variable
amount <- rep(c(1, .5, .2, .1, .05, .02, .01), each = 3)

# calculate amplification efficiency
pcr_efficiency(ct3,
               amount = amount,
               reference_gene = 'GAPDH')

# plot amplification efficiency
pcr_efficiency(ct3,
               amount = amount,
               reference_gene = 'GAPDH',
               plot = TRUE)

# }